The role of cell position and cell interactions in early mammalian embryogenisis will be studied in vitro, using clonal murine embryonal carcinoma (EC) cell lines as a model system. A hypothesis for the initial differentiation event, the formation of a "rind" of endoderm on the outside of an EC cell aggregate, will be revealed by mixing experiments involving pluripotent and variant nullipotent (unable to differentiate) EC cells. Nullipotent (F-9 or Nulli-SSC1) and pluripotent (PSA-1) EC cells will be mixed to construct aggregates with specific geometries. The developmental fate (endoderm, mesoderm, ectoderm, EC or death) of the two cell types will be followed by autoradiography in histological sections. To accomplish this, subclones of pluripotent and nullipotent cells that differ in their ability to incorporate 3H hypoxanthine will be used. These studies will test the hypothesis that cells must be located on the outside of an aggregate in order to differentiate into endoderm. The hypothesis suggested by the first set of experiments can be further tested by similar experiments in which endodermal cells are aggregated with pluripotent and nullipotent EC cells. For future research aimed at detailing the steps in the differentiation pathway, developmental mutants of pluripotent EC cells will be isolated. These studies will help to define the mechanisms that lead to the differentiation and consequently, the reversion of the malignant phenotype of EC cells.